There is a great need for the development of efficient and accurate methods related to the detection and identification of chemical and biological agents (hereinafter “analyte”) including, but not limited to, nucleic acids, proteins, illicit drugs, explosives, toxins, pharmaceuticals, carcinogens, poisons, allergens, and infectious agents. Current methods of detecting analytes require extraction of a sample into organic solvents, followed by analysis using stand alone analytical systems such as gas-liquid chromatography and/or mass spectroscopy. These methods are time-consuming and often expensive. The development of a biosensor device that could accurately and efficiently detect/screen for chemical and biological agents would therefore provide a significant cost and time benefit.
Three recent advancements in medicine are particularly germane to expanding the potential of detecting analytes, in particular with regard to the diagnosis and treatment of disease: nanotechnology, biodetectors (biosensors), and the identification of biomarkers for specific diseases and/or conditions. Nanotechnology, in particular nanoparticles, offers many advantages when used for applications such as the delivery of bioactive agents (i.e., DNA, AIDS drugs, gene therapy, immunosuppressants, chemotherapeutics), and drug uptake and degradation (i.e., enzyme encapsulation). For example, nanoparticles have been proposed as providing site-specific distribution of drugs to, and minimization of loss from, a target site. Appropriately sized particles have been proposed wherein such particles can be delivered to selected tissues to release their drug load in a controlled and sustained manner.
The term “biodetectors” or “biosensors” relates to the use of naturally occurring and synthetic compounds as highly specific and extraordinarily sensitive detectors of various types of molecules and markers of disease. Biosensor manufacture mimics the naturally occurring mechanisms of DNA, RNA, and protein synthesis in cells.
Aptamers have recently been identified as potentially effective biosensors for molecules and compounds of scientific and commercial interest (see Brody, E. N. and L. Gold, “Aptamers as therapeutic and diagnostic agents,” J. Biotechnol., 74(1):5–13 (2000) and Brody et al., “The use of aptamers in large arrays for molecular diagnostics,” Mol. Diagn., 4(4):381–8 (1999)). For example, aptamers have demonstrated greater specificity and robustness than antibody-based diagnostic technologies. In contrast to antibodies, whose identification and production completely rest on animals and/or cultured cells, both the identification and production of aptamers takes place in vitro without any requirement for animals or cells. Aptamer synthesis is far cheaper and reproducible than antibody-based diagnostic tests. Aptamers are produced by solid phase chemical synthesis, an accurate and reproducible process with consistency among production batches. An aptamer can be produced in large quantities by polymerase chain reaction (PCR) and once the sequence is known, can be assembled from individual naturally occurring nucleotides and/or synthetic nucleotides. Aptamers are stable to long-term storage at room temperature, and, if denatured, aptamers can easily be renatured, a feature not shared by antibodies. Furthermore, aptamers have the potential to measure concentrations of ligand in orders of magnitude lower (parts per trillion or even quadrillion) than those antibody-based diagnostic tests. These inherent characteristics of aptamers make them attractive for diagnostic applications.
A number of “molecular beacons” (often fluorescence compounds) can be attached to aptamers to provide a means for signaling the presence of and quantifying a target analyte. For instance, an aptamer specific for cocaine has recently been synthesized (Stojanovic, M. N. et al., “Aptamer-based folding fluorescent sensor for cocaine,” J. Am. Chem. Soc., 123(21):4928:31 (2001)). A fluorescence beacon, which quenches when cocaine is reversibly bound to the aptamer is used with a photodetector to quantify the concentration of cocaine present. Aptamer-based biosensors can be used repeatedly, in contrast to antibody-based tests that can be used only once.
Of particular interest as a beacon are amplifying fluorescent polymers (AFP). AFPs with a high specificity to TNT and DNT have been developed. Interestingly, a detector based on AFP technology also detects propofol, an intravenous anesthetic agent, in extremely low concentration. The combination of AFP and aptamer technologies holds the promise of robust, reusable biosensors that can detect compounds in minute concentrations with high specificity.
The term “biomarker” refers to a specific biochemical in the body that has a particular molecular feature to make it useful for diagnosing and measuring the progress of disease or the effects of treatment. For example, common metabolites or biomarkers found in a person's breath, and the respective diagnostic condition of the person providing such metabolite include, but are not limited to, acetaldehyde (source: ethanol, X-threonine; diagnosis: intoxication), acetone (source: acetoacetate; diagnosis: diet/diabetes), ammonia (source: deamination of amino acids; diagnosis: uremia and liver disease), CO (carbon monoxide) (source: CH2Cl2, elevated % COHb; diagnosis: indoor air pollution), chloroform (source: halogenated compounds), dichlorobenzene (source: halogenated compounds), diethylamine (source: choline; diagnosis: intestinal bacterial overgrowth), H (hydrogen) (source: intestines; diagnosis: lactose intolerance), isoprene (source: fatty acid; diagnosis: metabolic stress), methanethiol (source: methionine; diagnosis: intestinal bacterial overgrowth), methylethylketone (source: fatty acid; diagnosis: indoor air pollution/diet), O-toluidine (source: carcinoma metabolite; diagnosis: bronchogenic carcinoma), pentane sulfides and sulfides (source: lipid peroxidation; diagnosis: myocardial infarction), H2S (source: metabolism; diagnosis: periodontal disease/ovulation), MeS (source: metabolism; diagnosis: cirrhosis), and Me2S (source: infection; diagnosis: trench mouth).
Mechanisms of drug metabolism are extremely complex and are influenced by a number of factors including competitive binding on protein and red blood cells with other molecules, enzymatic activity, particularly in the liver, protein, and red blood cell concentration and a myriad of other factors. Exhaled breath holds the promise of a diagnostic technique, which can measure drug concentration real-time and thereby allow convenient determination of pharmacokinetics and pharmacodynamics of multiple compounds in real-time.
Accordingly, there are a number of medical conditions that can be monitored by detecting and/or measuring biomarkers present in a person's breath and other bodily fluids. While there has been technology generated towards the synthesis and use of aptamers and other multimolecular devices as biosensors, there exists little technology that address the use of exhaled breath in conjunction with apatmers as biosensors for the diagnosis and treatment of disease or as detectors for a wide range of naturally occurring and synthetic compounds. It is therefore desirable to provide a low-cost means for accurately and timely detecting and/or measuring the presence of metabolites in a person's bodily fluids in low concentrations via non-invasive methods. Further, in order to effectively apply exhaled breath sensing technology, there is a pressing need for an efficient screening method for determining which analytes/biomarkers are likely to be detectable in exhaled breath.